This invention relates to methods of crosslinking proteins, such as collagen, using peroxidase enzymes, such as horseradish peroxidase, in situ, to form biocompatible crosslinked semi-solid gels useful in a number of in vivo and in vitro applications, from wound healing, to drug delivery, to food processing.
Various collagen powders, sponges or other artificial constructs including xe2x80x9cartificial tissuexe2x80x9d or organs for medical uses have been described in the literature and in patents. All of these materials have been prepared ex situ, crosslinked or not, for later application to wounds. The same may be said for the use of collagen for augmentation of processed foods (meats, poultry). The inventors are not aware of any currently available products using collagen, or collagen used in combination with other co-reactants or additives, that can be prepared as a solution and applied by injection or pouring of a liquid at the site of interest (wound tissue or chopped meat or poultry), where covalent polymerization and bonding to the site is accomplished in situ. The closest known such wound sealants are polymers of purified fibrinogen (Sierra, 1993), crosslinked by addition of thrombin, and cyanoacrylate cements. The former represent the natural plasma proteins, in purified form, involved in clot formation. For human use they must be prepared from human plasma due to immunological considerations, and are used primarily in Europe and Asia (e.g., Japan), but have not been approved by the FDA (as of 1996) for U.S. use. Cyanoacrylate cements are more commonly known, for household use, as xe2x80x98supergluexe2x80x99. Both the fibrin and cyanoacrylate wound sealants are used primarily as alternatives to sutures (i.e., to seal surgical wounds), and have the disadvantage of forming such a dense polymer as to be only slowly biodegradable, thus often retarding wound remodeling and repair (Lasa et al., 1993). Neither is suitable for filling of larger wound beds or for use as a delivery vehicle for other components. Minor soft tissue wounds normally are capable of good repair with minimal treatment. More serious injuries (e.g., trauma), recalcitrant wounds (e.g., decubitus ulcers) or tissue repair which is compromised by other factors (e.g., age, diabetes) require assistance to heal. Healing of such wounds often is further complicated by infection, and where repair is finally achieved it occurs via scarring and contracture, usually resulting in some loss of cosmetic appearance and/or function. Attempts to improve repair may be made by mechanical means, where wound strength and barrier function are aided by a bioinert material (e.g., bandage) or by a biologic approach (e.g., resorbable sponges, grafts, xe2x80x9cartificial skinxe2x80x9d) which encourages regenerative repair. The former materials eventually must be removed, while materials for the latter often are deficient in mechanical strength, elasticity, or availability, are immunogenic or cytotoxic, or are expensive.
A similar adhesive for food use is Fibrimex(copyright), distributed in North America by F.N.A. Foods, Inc. It also is based on fibrinogen-thrombin reaction (materials obtained from bovine plasma) and will react at low temperature (4xc2x0 C.), but must be mixed very quickly with the meat products being reformed since the reaction proceeds to completion in less than 30 minutes.
We have demonstrated the feasibility of a new collagen-based wound sealant which can be gently but covalently crosslinked to a wound bed via a catalyzed chemical reaction utilizing peroxidase enzyme (primarily, horseradish peroxidase) and hydrogen peroxide (H2O2). Unlike other collagen compositions that are prepared ex situ, this material is prepared as a thick liquid that is poured or injected into the site of interest immediately upon activation by the catalyst, whereupon it polymerizes in situ. Good mechanical strength and elasticity by the polymer is achieved, cellular compatibility and faster healing is demonstrated, and the sealant can be prepared at relatively low cost. In addition, the base material could be modified by inclusion of other matrix components, co-reactants, non-reactive materials, growth factors, antibiotics and microbeads (e.g., of potential use for delayed-release of additional components). This material is particularly useful for emergency wound repair (e.g., use xe2x80x98in the fieldxe2x80x99), for repair of recalcitrant wounds (e.g., decubitus or pressure ulcers), and long-term regenerative type repair of a wound. Additional uses for this material include a slow release depot for vaccines, adjuvants or drugs, bone repair, graft or prosthetic implant stabilization, and as a binding agent for restructured foods (e.g., sausages, xe2x80x98poultry rollsxe2x80x99, restructured meats). This material thus represents a new product and composition of matter as well as a potential improvement of existing products and compositions currently available.
This material has use throughout veterinary medical, human medical and dental practice, and the food industry.
Collagen may be crosslinked by a variety of chemical or physical means (for reviews, see Rault et al., 1996; Jain, 1992; Meade and Silver, 1990) and the products subsequently used (crosslinked or not) in various wound repair materials (Pachence, 1996; Choucair and Phillips, 1996; Jeter and Tintle, 1991). While these products may have acceptable mechanical strength, antigenicity and permeability to cellular ingrowth and remodeling, none of the crosslinking methods are compatible with living cells and therefore cannot be employed in situ. Crosslinking of collagen solutions into gels using lactoperoxidase (Tenovuo and Paunio, 1979) or horseradish peroxidase ( LaBella et al., 1968) has been demonstrated and the formation of small amounts of dityrosine reported, however, no use for these collagen gels was demonstrated or suggested.
We have explored the use of peroxide-peroxidase catalyzed crosslinking of collagen with the aim of developing one or more compositions or materials that may be used by the medical, pharmaceutical or food industries. The following are embodiments of the present invention.
One embodiment relates to a method of covalent crosslinking of acid-soluble Type I collagen from calf skin with horseradish peroxidase (HRP)-hydrogen peroxide (H2O2, O2, Px) (collagen polymer). Collagen solutions of 8-12 mg/ml can optimally be crosslinked into a semi-solid gel by addition of HRP and peroxide in molar ratios of 4-5:1:50 to 4-5:1:200 collagen:HRP:peroxide (molecular weight basis, e.g., moles of each component). Controls lacking either HRP or peroxide failed to form a gel. Greater ratios of collagen relative to HRP (e.g., 20:1:200) or substantially higher concentrations of peroxide ( greater than 400 parts) did not provide optimal polymerization.
That a crosslinked polymer was being formed was confirmed by decreased solubility of the matrices in hot (75xc2x0 C.) sodium dodecyl sulfate (SDS) solution, and an increase in high molecular weight components observed by SDS agarose gel filtration and SDS-polyacrylamide gel electrophoresis with a concomitant decrease in lower molecular weight components (monomers).
The matrix was found to have significant mechanical strength and elasticity by Instron compression analysis and deformation testing, and was superior in these respects to glutaraldehyde crosslinked positive control preparations. Uncrosslinked materials had no demonstrable mechanical strength as measured by either method.
Other embodiments relate to the crosslinking of interstitial collagen types II, III and of type IV collagen (basement membrane type) in the presence of HRP: peroxide, therefore the method is useful for both interstitial and basement membrane collagens.
One embodiment relates to the solidification (crosslinking) of solutions of type I collagen using soybean peroxidase (Sigma(copyright)) and microbial peroxidase (Sigma(copyright), from Arthromyces ramosus), although to a lesser apparent degree than with HRP. The method thus appears to be generally applicable to the use of any peroxidase.
Other embodiments cover the fact that other proteins (i.e., fibrinogen, recombinant fibronectin-like engineered protein polymer, Sigma(copyright)), alone or in combination with type I collagen, can be crosslinked in this system.
Another embodiment relates to a co-polymer of type I collagen and fibrinogen, resulting in greater mechanical strength than an equal amount of collagen alone.
Another embodiment relates to the fact that some proteins (e.g., bovine serum albumin, BSA) apparently are not crosslinked in the peroxide:peroxidase system. This protein could, however, be incorporated into collagen gels (1 mg BSA per 10 mg/ml collagen type I, i.e., 10 weight percent relative to collagen) without interfering appreciably with the crosslinking of the collagen. Thus, the matrix can be useful as a depot for the delivery of materials which do not participate in or are altered by the action of HRP:peroxide (e.g., for vaccines, adjuvants, growth factors, drugs).
Another embodiment relates to microbeads, using Sephadex G25, fine beads as a model system, can be incorporated into the collagen polymer matrix by at least 10 weight percent with no apparent effect on the mechanical strength or integrity of the matrix. Thus, materials incorporated into (biodegradable) microbeads may potentially be incorporated into the matrix for timed (delayed) release of entrapped components (e.g., vaccines, adjuvants, growth factors, drugs).
Further, in another embodiment, the polymer is useful as a three dimensional matrix for the growth of dermal fibroblasts (e.g., mouse 3T3 cells) in vitro. Cells readily grow into, proliferate in and remodel the matrix without signs of any cytotoxicity.
A still further embodiment relates to the fact that the collagen matrix can be modified by the addition of recombinant fibronectin polymer (Fn) and/or human placental hyaluronic acid (HA)(Sigma(copyright)). An optimal addition of 0.5% Fn and 1% HA was found in vitro to significantly encourage cell ingrowth, proliferation and protein synthesis as determined by labeling of 3T3 cells with 3H-thymidine and 14C-proline, over a period of one week. Histological observations confirmed the radioisotopic incorporation results.
In another embodiment, recombinant human basic fibroblast growth factor (rhbFGF) can be added to the collagen matrix prior to polymerization, or to the matrix plus Fn/HA, and will produce increased cellular proliferation and protein synthesis above that seen with the base collagen matrix or Fn/HA modified matrix alone (control matrices) over a period of one week. Thus, growth factors/cytokines may be added to the matrix and retain effective cell stimulating activity above and beyond that of the matrix polymer components alone.
Still further, repeated in vivo applications of the polymer in rats and mice indicate that the collagen polymer is weakly or non-immunogenic. Subcutaneous injection of the polymer in mice persists for at least 5 days to 2 weeks, further indicating its potential for use as a delivery depot. No inflammatory reaction, upon histological examination, was found after four exposures to the polymer over an eight week period indicating that the material is safe for repeated exposure.
Furthermore, in another embodiment it has been shown that the collagen polymer improves healing of granulation-type wounds in rats by, at minimum, decreasing initial wound expansion. Decreased scar formation also may result, although this was not quantified.
Still further, it has been shown that Fn/HA modified matrix significantly improves healing of granulation-type wounds in rats, and that addition of rhbFGF to the modified matrix results in even greater improvement of healing by, at minimum, decreasing initial wound expansion. Decreased scar formation also may result.
Additionally, in another embodiment, it has been demonstrated that the matrix has an inhibitory effect on the growth of Staphylococcus aureus in agar plate culture when the peroxide was included in the mixture. Further inhibition of bacterial growth was achieved by inclusion of a xe2x80x9cmodelxe2x80x9d antibiotic (gentamicin) in the polymer. Polymer formation was inhibited by direct inclusion of the antibiotic into the collagen matrix, suggesting co-reaction which resulted in interference with the crosslinking reaction of the collagen. It is suggested that antibiotics or other appropriate drugs or other bioactive compounds can be incorporated into biodegradable microbeads for delayed release, and that these beads can be incorporated into the collagen matrix essentially as stated above. This delivery system would be a more effective means of inclusion of such materials into the collagen-based matrix as a method of avoiding participation in the crosslinking reaction initiated by peroxide:peroxidase.
It has further been demonstrated that in embodiments where dilute solutions of type I collagen are reacted with a molar excess of either phosposerine or phosphoarginine in the presence of HRP-peroxide, the apparent molecular weight of the xcex11, and xcex12 chains, after separation by SDS-PAGE, increases slightly. The increase suggests a covalent binding of the phospho-amino acids in ratios of about 2.5-3.5 residues per collagen chain. It is suggested that the collagen chains thus can be directly modified by addition of other components via HRP-peroxide catalyzed reaction.
Still further it has been demonstrated that in some embodiments the collagen polymer may be mixed with or applied to the surface of meat and poultry tissues for use as a food binding/restructuring agent. The liquid nature of the starting material allows thorough blending with the minced or chopped meat or poultry via mechanical mixing, and will polymerize over a period of hours at 4xc2x0 C. The restructured products maintain a superior degree of cohesiveness and tenderness after cooking to USDA approved endpoint temperatures. All components of the polymer are food grade and acceptable for incorporation into meat products. The antimicrobial activity of the reactants described above may have food safety implications for the food industry. The addition of other materials (e.g., alkaline phosphates, sodium chloride) to the collagen polymer also can augment its structural/biomechanical properties in this application.
An embodiment of the invention, the collagen polymer, with or without additional additives or the addition of phospo-amino acids, also may provide a matrix material suitable for stimulation of mineralization and which might be useful for bone repair and for fixation of orthopedic (e.g., hip replacements) or dental implants in bone.
An embodiment of the invention, the collagen polymer, with or without additional additives, also may be useful for graft augmentation (e.g., skin grafts), to augment healing of surgical injuries, or for cosmetic injection procedures.
The use of peroxide with peroxidase (primarily HRP) to gel and apparently crosslink and polymerize collagen (primarily acid soluble type I) has been demonstrated. Assessments of mechanical strength and elasticity have been made and compared to appropriate positive and negative controls. The ability to add other components to the collagen matrix has been demonstrated. The ability of skin fibroblasts to grow in vitro into the matrix with significant proliferation and synthetic activities has been shown. Formation of the collagen polymer in vivo in granulating wounds or subcutaneous injections in rats and mice, with subsequent persistence at the application site, has been demonstrated. A lack of inflammatory reaction to the collagen matrix, combined with its in vivo resorption and apparent ability to facilitate wound closure, was observed. The ability of the catalyzed collagen matrix to bind meat particles together in a useful fashion for restructuring of meat/poultry products has been demonstrated.
Non-limiting examples of advantages of the present invention include, but are not limited to, the following:
The material is easily and cheaply prepared for medical use, relative to current wound repair materials.
It is readily shaped to fit where needed since it is applied as a liquid.
It is tissue-compatible for covalent crosslinking in situ, thus binding to the wound bed as well as to itself.
It uses no toxic chemicals at any point in its preparation, thus is less likely to cause an inflammatory reaction than, e.g., glutaraldehyde crosslinked collagen.
It does not have to be rehydrated, like dry collagen preparations.
It retains a high degree of elasticity along with a reasonable mechanical strength, and thus can stretch within a wound bed without tearing or pulling excessively on the margins of the wound.
The polymer has inherent antimicrobial activity (which can be enhanced by added antibiotics), thus can reduce the incidence and severity of later complications when applied to contaminated wounds.
It is readily invaded by cells and degradable for remodeling and replacement (unlike fibrinogen or cyanoacrylate glues).
The matrix can be readily modified by addition of other components/co-reactants, thus having the potential to be tailored to a particular application.
The material initially is injectable and persists long enough to be useful as a slow release depot, e.g., for vaccines, adjuvants, drugs or other bioactive materials.
The material also will accommodate a reasonable load of microbeads, and thus can deliver materials that are initially protected from the peroxide-peroxidase reaction.
The addition of bioactive components to the collagen matrix, along with additional components contained in biodegradable microbeads, provides a mechanism for the sequential delayed release of two or more components (xe2x80x9ctime release-time releasexe2x80x99).
The matrix can be readily mixed with meats and poultry and reacted at low temperature (e.g., 4xc2x0 C.) to contribute strength and cohesiveness to the food product. It also has been shown to withstand cooking temperatures of at least 71xc2x0 C. while still making significant contributions to the food products.
Where a protein is referred to, in the specification and claims, it is intended that the coverage refers to wild type protein, naturally or recombinantly expressed, as well as to derivatives of the protein, including, but not limited to, deletions, insertions, modifications and the like known in the art.
Other and further objects, features and advantages would be apparent and eventually more readily understood by reading the following specification and by reference to the company drawing forming a part thereof, or any examples of the presently preferred embodiments of the invention are given for the purpose of the disclosure.